In some cases it is desirable to control the time or extent of expression of a phenotypic trait in plants, plant cells or plant tissue. An ideal situation would be the regulation of expression of such a trait at will, triggered by a chemical that could be easily applied to field crops, ornamental shrubs, etc. One such system of regulating gene expression which could be used to achieve this ideal situation, as yet unknown to be present naturally in plants, is the steroid and thyroid hormone superfamily of nuclear receptors.
The steroid and thyroid hormone superfamily of nuclear receptors is found in mammals and insects and is composed of over 100 known proteins. These receptors fall into at least two functionally distinct categories known as Class I and Class II. Beato, Cell 56: 335-344(1989); Parker, Sem. Cancer Biol. Ser. 1: 81-87(1990). Of the two classes, only the Class II receptors function in the nucleus as heterodimers to affect expression of target genes in the presence of hormone. The best studied examples of Class II receptor proteins are Retinoic Acid Receptor (RAR), Vitamin D Receptor (VDR), and Thyroid Hormone Receptor (T3R) and Retinoic X Receptor (RXR). The receptors bind to the 5′ regulatory region of the target gene and, upon binding of a chemical ligand to the receptor, the transcriptional activation (transactivation) domain of the receptor affects gene expression by interacting with other transcription initiating factors.
In addition to the Class II receptor proteins found in mammals as described above, receptors of similar structure and activity have been indentified in the insect Drosophila. Koelle et al., Cell 67: 59 (1991); Christianson and Kafatos, Biochem. Biophys. Res. Comm. 193: 1318 (1993); Henrich et al., Nucleic Acids Res. 18: 4143 (1990). The Ecdysone Receptor (EcR) binds the steroid hormone 20-hydroxyecdysone and, when heterodimerized with the product of the Ultraspiracle gene (USP), will transactivate gene expression. USP is most homologous to RXRα, and RXR is capable of forming heterodimers with EcR. Thomas et al., Nature 362: 471-475 (1993). Additional chemical ligands besides 20-hydroxyecdysone, such as other hormone agonists or antagonists, will also bind to these receptors and cause transactivation of a target gene.
One member of the steroid and thyroid superfamily of nuclear receptors, the Class I Glucocorticoid Receptor (GR) which utilizes chaperonins and does not function by heterodimerization with other receptors, has been shown to transactivate a target gene in plant cells. Schena et al., Proc. Natl. Acad. Sci. USA 88: 10421-10425 (1991). A fragment containing the ligand binding domain from GR was fused to the anthocyanin regulatory protein known as ‘R’ and shown to stimulate production of anthocyanin in transgenic Arabidopsis thaliana in response to the application of the appropriate chemical ligand. Lloyd et al., Science 226: 436 (1994). It was also reported by Lloyd et al. that full-length GR did not activate gene expression in stably transformed Arabidopsis thaliana whereas it did in transient assays in tobacco protoplasts. Furthermore, fusions of R with a fragment from the Estrogen Receptor (ER), another Class I receptor which utilizes chaperonins, also stimulated production of anthocyanin in the presence of the appropriate chemical ligand but showed ‘substantial’ background expression.
The distinguishing feature of the Class II receptor proteins, transactivation of a target gene by heterodimerized receptors in the presence of an appropriate chemical ligand, offer previously unrecognized opportunities for chemical control of gene expression in plants. The use of heterodimers allows a broader range of gene control strategies, and chemicals are already known for agricultural use which can trigger receptor-mediated transactivation of target gene expression of this class. Furthermore, gene control strategies for plants which utilize nuclear receptors that do not occur naturally in plants have the attractive feature of inducing only the genetically engineered target gene. The class II receptors in general, however, possess fairly poor transcriptional activation domains, and the ability of the receptors to transactivate target genes may be enhanced by the addition of other transcriptional activation domains, particularly from plant or viral species. Further modification would also be needed in order to provide minimum basal activity which increases rapidly to high levels in the presence of a triggering chemical. As has been demonstrated by the present invention, receptor polypeptides based on the class II model, and the genes that encode them, have been developed which function in plant cells to control expression of a target polypeptide wherein the receptor polypeptides activate the 5′ regulatory region of a target expression cassette in the presence of a chemical ligand. Such a method of controlling gene expression in plants would be useful for controlling various traits of agronomic importance, such as plant fertility.